Cationic lipids with various head groups for oligonucleotide delivery

ABSTRACT

The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with siRNA, to facilitate the cellular uptake and endosomal escape, and to knockdown target mRNA both in vitro and in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/390,702 filed Feb. 16, 2012, now U.S. Pat. No. 9,181,295, issuedon Nov. 10, 2015, which is a 35 U.S.C. §371 National Phase EntryApplication of International Application No. PCT/US2010/045854 filedAug. 18, 2010, which designates the U.S., and which claims benefit under35 U.S.C. §119(e) of the U.S. Provisional Application No. 61/235,476,filed Aug. 20, 2009, and U.S. Provisional Application No. 61/345,754,filed May 18, 2010, the contents of each of which are incorporatedherein by reference in their entirety

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is being submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “20151006_Sequence_Listing_from_Parent_051058-081171-C”,creation date of Oct. 6, 2015 and a size of 3.2 KB. This sequencelisting submitted via EFS-Web is part of the specification and is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to novel-cationic lipids that can be usedin-combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides; tofacilitate the cellular uptake and endosomal escape, and to knockdowntarget niRNA both in vitro and in vivo.

Cationic lipids and the use of cationic lipids in lipid nanoparticlesfor the delivery of oligonucleotides, in particular siRNA and miRNA,have been previously disclosed. Lipid nanoparticles and use of lipidnanoparticles for the delivery of oligonucleotides, in particular siRNAand miRNA, has been previously disclosed. Oligonucleotides (includingsiRNA and miRNA) and the synthesis of oligonucleotides hasbeen-previously disclosed. (See U.S. patent applications: US2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/127060,WO2009/132131, WO2010/042877, WO2010/054384, WO2010/054401,WO2010/054405 and WO2010/054406). See also Semple S. C. et al, Rationaldesign of cationic lipids for siRNA delivery, Nature Biotechnology,published online 17 January 2010; doi:10.1038/nbt. 1602.

Butyl CLinDMA is one of the cationic lipids disclosed in US2006/0240554. Other cationic lipids are also generically disclosed in US2006/0240554 including Octyl CLinDMA. We have synthesized and testedboth Butyl CLinDMA and Octyl CLinDMA in lipid nanoparticle formulationsand found that Octyl CLinDMA has superior properies to Butyl CLinDMA.Octyl CLinDMA (otherwise known as OCD) is also described inWO2010/021865.

We adopted a rational approach to design cationic lipids for use inlipid nanoparticle formulations to deliver small interfering RNA(siRNA). Starting with the cationic lipid Octyl CLinDMA (designated,“Compound 12” in this application) as a benchmark, we designed novelcationic lipids with superior properties as-demonstrated in mouse, ratand monkey experiments.

The compounds of the instant invention contain a 1,3-diether linker.

It is an object of the instant invention to provide novel cationiclipids that can be used in combination with other lipid components suchas cholesterol and PEG-lipids to form lipid nanoparticles witholigonucleotides, to facilitate the cellular uptake and endosomalescape, and to knockdown target mRNA both in vitro and in vivo.

SUMMARY OF THE INVENTION

The instant invention provides for novel cationic lipids that can beused in combination with other lipid components such as cholesterol-andPEG-lipids to form lipid nanoparticles with oligonucleotides, tofacilitate the cellular uptake and endosomal escape, and to knockdowntarget mRNA both in vitro and in vivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Mouse in vivo Cytokine Induction 3 hour post injection.

FIG. 2: Mouse data comparing Compound 12 with Compound 9.

FIG. 3: Rat data comparing Compound 12 with Compound 9.

FIG. 4: Monkey data comparing Compound 12 with Compound 9.

DETAILED DESCRIPTION OF THE INVENTION

The various aspects and embodiments of the invention are directed to theutility of novel cationic lipids useful in lipid nanoparticles todeliver oligonncleotides, in particular, siRNA and miRNA, to any targetgene. (See U.S. patent applications: US 2006/0083780, US 2006/0240554,US 2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patentapplications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 andWO2010/054406). See also Semple S. C. et al., Rational design ofcationic lipids for siRNA delivery. Nature Biotechnology, publishedonline 17 Jan. 2010; doi: 10.1038/nbt. 1602.

The cationic lipids of the instant invention are useful components in alipid nanoparticle for the delivery of oligonucleotides, specificallysiRNA and miRNA.

In a first embodiment of this invention, the cationic lipids areillustrated by the Formula A:

wherein:

p is 1 to 8;

R¹ and R² are independently selected from H, (C₁-C₁₀)alkyl,heterocyclyl, and a polyamine, wherein said heterocyclyl and polyamineare optionally substituted with one to three substituents selected fromR⁴, or R¹ and R² can be taken together with the nitrogen to which theyare attached to form a monocyclic heterocycle with 4-7 membersoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle optionally substituted with one to three substituentsselected from R⁴;

R³ is selected from H and (C₁-C₆)alkyl, said alkyl optionallysubstituted with one to three substituents selected from R⁴;

R⁴ is independently selected from halogen, OR⁵, SR⁵, CN, CO₂R⁵ andCON(R⁵)₂;

R⁵ is independently selected from H, (C₁-C₁₀)alkyl and aryl; and

Y is a(C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl, or a (C₄-C₂₂)alkenyl;

or any pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the invention features a compound having FormulaA, wherein;

p is 1 to 8;

is selected from:

n is 1 to 10;

R³ is H; and

Y is a (C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl, or a (C₄-C₂₂)alkenyl;

or any pharmaceutically acceptable salt or stereoisomer thereof.

Specific cationic lipids are:

-   (2 S)-1-{7-[(3 β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4    Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine (Compound 4);-   (2 R)-1-{4-[(3 β)-cholest-5-en-3-yloxy]butoxy}-3-[(4    Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine (Compound 5);-   1-[(2 R)-1-{4-[(3    β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine    (Compound 6);-   1-[(2 R)-1-{7-[(3    β)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9 Z, 12    Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine (Compound 7);-   1-[(2 R)-1-{4-[(3 β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9    Z, 12 Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine (Compound 8);-   (2S)-1-({6-[(3β)-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine    (Compound 9);-   (3β)3-[6-{[(2S)-3-[(9Z)octadec-9-en-1-yloxyl]-2-(pyrrolidin-1-yl)propyl]oxy}hexyl)oxy]cholest-5-ene    (Compound 10);-   (2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy))propan-2-amine    (Compound 11);-   (2R)-1-({8-[(3β)cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine    (Compound 13);-   (2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3(heptyloxy)-N,N-dimethylpropan-2-amine    (Compound 14);-   (2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1-yloxy]propan-2-amine    (Compound 15);-   (2S)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine    (Compound 16);-   (2S-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-[2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,-hexadecafluorononyl)oxy]-N,N-dimethylpropan-2-amine    (Compound 17);-   2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]nonyl}oxy)-2-    {[(9Z,12Z)-octadeca-9,12-dine-1-yloxy]methyl}propan-1ol (Compound    19); and;-   2-amino-3-({6-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5en-3-yloxy]hexyl}oxy)-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol    (Compound 20);    or any pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the cationic lipids disclosed are useful in thepreparation of lipid nanoparticles.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nonoparticle for the delivery of oligonucleotides.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA and miRNA.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA.

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiRNA and miRNA:

-   Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;-   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;-   Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;-   Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;-   Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;-   Cationic Lipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10; and-   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10.

The cationic lipids of the present invention may have asymmetriccenters, chiral axes, and chiral planes (as described in: E. L. Elieland S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons,New York, 1994, pages 1119-1190), and occur as racemates, racemicmixtures, and as individual diastereomers, with all possible isomers andmixtures thereof, including optical isomers, being included in thepresent invention. In addition, the cationic lipids disclosed herein mayexist as tautomers and both tautomeric forms are intended to beencompassed by the scope of the invention, even though only onetautomeric structure is depicted.

When any variable (e.g. R⁴) occurs more than one time in anyconstituent, its definition on each occurrence is independent at everyother occurrence. Also, combinations of substituents and variables arepermissible only if such combinations result in stable compounds.

If the ring system is bicyclic, it is intended that the bond be attachedto any of the suitable atoms on either ring of the bicyclic moiety.

It is understood that substituents and substitution patterns on thecationic lipids of the instant invention can be selected by one ofordinary skill in the art to provide cationic lipids that are chemicallystable and that can be readily synthesized by techniques known in theart, as well as those methods set forth below, from readily availablestarting materials. If a substituent is itself substituted with morethan one group, it is understood that these multiple groups may be onthe same carbon or on different carbons, so long as a stable structureresults.

It is understood that one or more Si atoms can be incorporated into thecationic lipids of the instant invention by one of ordinary skill in theart to provide cationic lipids that are chemically stable and that canbe readily synthesized by techniques known in the art from readilyavailable starting materials.

In the compounds of Formula A, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of Formula A. Forexample, different isotopic forms of hydrogen (H) include protium (¹H)and deuterium (²H). Protium is the predominant hydrogen isotope found innature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples. Isotopically-enriched compoundswithin Formula A can be prepared without undue experimentation byconventional techniques well known to those skilled in the art or byprocesses analogous to those described in the Scheme and Examples hereinusing appropriate isotopically-enriched reagents and/or intermediates.

As used, herein, “alkyl” means a saturated aliphatic hydrocarbon havingthe specified number of carbon atoms.

As used herein, “alkenyl” means an unsaturated aliphatic hydrocarbonhaving the specified number of carbon atoms.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 atoms in each ring, wherein at least onering is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydro-naphthyl, indanyl and biphenyl.

As used herein, “heterocyclyl” means a 4- to 10-membered aromatic ornonaromatic heterocycle containing from 1 to 4 heteroatoms selected fromthe group consisting of O, N and S, and includes bicyclic groups.“Heterocyclyl” therefore includes, the following: benzoimidazolyl,benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl,benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl,furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl,isobenzofuranyl, isomdolyl, isoquinolyl, isothiazolyl, isoxazolyl,naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline,oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl,pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl,thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, pipcridinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof all of which are optionally substituted with one tothree substituents selected from R³.

As used herein, “polyamine” means compounds having two or more primaryamino groups—such as putrescine, cadaverine, spermidine, and spermine.

In an embodiment,

is selected from:

In an embodiment,

is selected from:

In an embodiment,

is selected from NH₂ and NMe₂.

In an embodiment,

is NH₂.

In an embodiment, n is 1 to 5.

In an embodiment, p is 1 to 8.

In an embodiment, R³ is H or (C₁-C₆)alkyl, said alkyl is optionallysubstituted with from one to three OH.

In an embodiment, R³ is H, methyl, ethyl or propyl, said methyl, ethylor propyl is optionally substituted with one OH.

In an embodiment, R³ is H or hydroxymethyl.

In an embodiment, R³ is H.

In an embodiment, R⁴ is independently halogen, OH, O(C₁-C₆)alkyl, SH,S(C₁-C₆)alkyl, CN, CO₂H, CO₂(C₁-C₆)alkyl, CONH₂, or CON(C₁-C₆)alkyl₂.

In an embodiment, R⁴ is independently halogen, OH or O(C₁-C₆)alkyl.

In an embodiment, R⁵ is independently H or (C₁-C₆)alkyl.

In an embodiment, Y is a (C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl, ora(C₄-C₂₂)alkenyl.

In an embodiment of Formula A, “heterocyclyl” is pyrolidine, piperidine,morpholine, imidazole or piperazine.

In an embodiment of Formula A, “monocyclic heterocyclyl” is pyrolidine,piperidine, morpholine, imidazole or piperazine.

In an embodiment of Formula A, “polyamine” is putrescine, cadaverine,spermidine or spermine.

Included in the instant invention is the free form of cationic lipids ofFormula A, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific cationic lipidsexemplified herein are the protonated salts of amine cationic lipids.The term “free form” refers to the amine cationic lipids in non-saltform. The encompassed pharmaceutically acceptable salts not only includethe isolated salts exemplified for the specific cationic lipidsdescribed herein, but also all the typical pharmaceutically acceptablesalts of the free form of cationic lipids of Formula A. The free form ofthe specific salt cationic lipids described may be isolated usingtechniques known in the art. For example, the free form may beregenerated by treating the salt with a suitable dilute aqueous basesolution such as dilute aqueous NaOH, potassium carbonate, ammonia andsodium bicarbonate. The free forms may differ from their respective saltforms somewhat in certain physical properties, such as solubility inpolar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant cationic lipids canbe synthesized from the cationic lipids of this invention which containa basic or acidic moiety by conventional chemical methods. Generally,the salts of the basic cationic lipids are prepared either by ionexchange chromatography or by reacting the free base with stoichiometricamounts or with an excess of the desired salt-forming inorganic ororganic acid in a suitable solvent or various combinations of solvents.Similarly, the salts of the acidic compounds are formed by reactionswith the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the cationic lipids of thisinvention include the conventional non-toxic salts of the cationiclipids of this invention as formed by reacting a basic instant cationiclipids with an inorganic or organic acid. For example, conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like, as well as salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,trifluoroacetic (TFA) and the like.

When the cationic lipids of the present invention are acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric; ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the cationic lipids of the present inventionare potentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

UTILITY

Cationic lipids and the use of cationic lipids in lipid nanoparticlesfor the delivery of oligonucleotides, in particular-siRNA and miRNA, fortherapeutic purposes, have been previously disclosed. (See U.S. patentapplications: US 2006/0240554 and US 2008/0020058). Lipid nanoparticlesand use of lipid nanoparticles for the delivery of oligonucleotides, inparticular siRNA and miRNA, for therapeutic purposes, has beenpreviously disclosed. (See U.S. patent applications: US 2006/0240554 andUS 2008/0020058). Oligonucleotides (including siRNA and miRNA) and thesynthesis of oligonucleotides has been previously disclosed. (See U.S.patent applications: US 2006/0240554 and US 2008/0020058).

The utility of cationic lipids in LNPs for the delivery ofoligonucleotides for therapeutic purposes is also disclosed in Guo, K.et al. Molecular Pharaceutics (2009) 6:3, 651-658; and Whitehead, K. A.et al. Nature Reviews Drug Discovery (2009) 8, 129-138.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof. The reagents utilized in synthesizingthe cationic lipids are either commercially available or are readilyprepared by one of ordinary skill in the art.

GENERAL SCHEMES

General Scheme 1: Synthesis of the novel cationic lipids is a convergentprocess whereby the Lewis acid catalyzed reaction between the epoxide(i) and the linker (ii) affords the desired alcohol regioisomer (iii).Functional group interconversion of the hydroxyl group to a suitableleaving group, for example a triflate group ((iv), X=OTf), followed bydisplacement with an appropriate amine affords the requisite cationiclipid (v).

General Scheme 2: Cationic lipids of type (x) can be prepared asoutlined below. Aminodiols can be protected as a cyclic hemi-aminal(vi). Alkylation of the free alcohol with linoleyl mesylate can generatecompounds of type (vii). Deprotection of the hemi-aminal followed byprotection of the primary amine can generate alcohol (vii). Alkylationof this alcohol, deprotection of the amine and reductive animation canyield compounds of type (x).

General Scheme 3: Compounds of type (xv) can be prepared as outlinedbelow. The amino group of tris can be protected and the diol can beconverted to a cyclic ketal. Alkylation of the free alcohol can generatecompound (xii). Deprotection of the ketal followed by amine reprotectioncan give diol (xiii). A synthetic scheme analogous to that described inGeneral Scheme 2 would then generate compounds of type (xv).

General Scheme 4: Compounds of type (xvii) can be prepared as outlinedbelow. 2-amino-2-(hydroxymethyl)propane-1,3-diol can be monoalkylatedupon treatment with sodium hydride and a lipid electrophile in tolueneto generate the mono-ether derivative (xvi). A second alkylation can beconducted under similar conditions to generate the diether trisderivatives of type (xvii).

Preparation of (2R)-2-{[4Z)-dec-4-en-1-yl]methyl}oxirane (Compound 1)

To a stirred, cooled (<5° C.) mixture of cis-4-decen-1-ol (1,14.42 g,156 mmol), tetrabutylammonium bromide (1.26 g, 3.91 mmol) and sodiumhydroxide (4.67 g, 117 mmol) was added R-(-)-epichlorohydrin (6.1 mL, 78mmol) in one portion, the mixture was stirred for 2 hours, and then asecond portion of R-(-)-epichlorohydrin (6.1 mL, 78 mmol) was added. Themixture was stirred overnight. Hexane (150 mL) was added, and themixture filtered. The filtrate concentrated to oil, and purificationthrough flash chromatography gave product (1, 14.3 g, 67.3 mmol) in 86%yield. Compound 1: C₁₃H₂₄O₂ HRMS M+H expected 213.1856; found 213.1849amu. ¹H NMR (500 MHz, CDCl₃) δ 5.34 (2H, multiplet); 3.70 (1H, dd,J=3.2, 11.5 Hz); 3.52 (1H, dt, J=6.6, 9.2 Hz); 3.47 (1H, dt, J=6.6, 9.2Hz); 3.39 (1H, dd, J=5.5, 11.6 Hz); 3.15 (1H, multiplet); 2.80 (1H, dd,J=4.1, 4.9 Hz); 2.61, (1H, dd, J=2.7, 5.0 Hz); 2.11 (2H, q, 7.2 Hz);2.02 (2H, q, J=7.2 Hz); 1.66 (1H, q, J=6.7 Hz); 1.63 (1H, t, J=6.9 Hz);1.38-1.24 (6H, complex); 0.89 (3H, t, J=6.9 Hz) ppm.

Preparation of 8-[(3β)-cholest-5-en-3-yloxy]octan-1-ol (Compound 2)

A mixture of 1,7-heptandiol (30.6 g, 231 mmol) and cholesteryl tosylate(25 g, 46.2-mmol) in toluene (80 mL) was heated at 80° C. for 16 hours.The reaction mixture was cooled to room temperature and hexane (70 mL)added. The resulting two layers were separated and the top layercollected and washed with a 1:1 solution of saturated brine and 1MNa₂CC₃ (100 mL), dried over Na₂SO₄ filtered, and finally concentrated tolow volume (˜40 mL). The crude oil was purified through flashchromatography to give product (2, 17.0 g, 33.9 mmol) in 73% yield.Compound 2: C₃₄H₆₀O₂: HRMS M+Na expected 523.4491; found 523.4486 amu.¹H NMR (500 MHz, CDCl₃) δ 5.34 (1H, complex); 3.64 (2H, complex); 3.45(2H, complex); 3.12 (1H, complex); 2.35 (1H, ddd, J=2.2, 4.7, 13.2 Hz);2.19 (1H, complex); 2.05-1.77 (5H, complex); 1.63-0.83 (44H, complex);0.68 (3H,s) ppm.

Preparation of (2 R)-1-[{7-[(3 β)-cholest-5-en-3-yloxy]heptyl}oxy)-3-[(4Z)-dec-4-en-1-yloxy]propan-2-ol. (Compound 3)

To a stirred, cooled (0° C.), solution of the alcohol (2, 6.00 g, 12mmol) in anhydrous CH₂Cl₂ (20 mL) under an atmosphere of nitrogen wasadded 1M SnCl₄ in CH₂Cl₂ (1.2 mL, 1.2 mmol). A solution of the epoxide(1, 3.1 g, 14.6 mmol) in CH₂Cl₂ (5 mL) was added dropwise over 2 hours.The solution was stirred for a further 1 hour. The reaction was quenchedwith 1 M Na₂CO₃ (3 mL) and hexane (50 mL) added. The mixture wasfiltered, and the filtrate collected and concentrated to a crude oil(9.7 g). Purification through flash chromatography afforded product (3,4.95 g, 6.94 mmol) in 58% yield. C₄₂H₈₄O₄ HRMS M+H expected 713.6442;found 713.6423 amu. ¹H NMR (500 MHz, CDCl₃) δ 5.42-5.30 (3H, complex);3.94 (1H, complex); 3.52-3.40 (10H, complex); 3.12 (1H, complex); 2.45(1H, d, J=4.1 Hz); 2.35 (1H, ddd, J=2.4, 13.2 Hz); 2.18 (1H, br t,J=12.7 Hz); 2.09 (2H, br q, J=7.0 Hz); 2.05-1.77 (7H, complex);1.67-0.82 (55H, complex); 0.68 (3H, s) ppm.

Preparation of (2 S )-1-{7-[(3 β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine. (Compound 4)

To a stirred cooled (0° C.) solution of the alcohol (3, 4.9g, 6.87 mmol)and anhydrous pyridine (0.7 mL, 8.67 mmol) in anhydrous CH₂Cl₂ (20 mL),under a nitrogen atmosphere, was added triflic anhydride (1.4 mL, 8.3mmol) dropwise. The solution was stirred for 2 hours, and then added toa 2M solution of dimethylamine in THF (18 mL, 36 mmol) at 0° C. Thesolution was stirred for 2 hours, and then dichloromethane (150 mL)added. The solution was then washed with 0.5 M NaHCO₃ (150 mL). Theorganic layer was separated and dried over sodium sulfate, filtered andconcentrated to oil (6.2 g). Hexane (60 mL) was added, the solids formedremoved by filtration and the filtrate concentrated to give a crude oil(5.4 g). Purification through chromatography afforded product (4, 2.83g, 3.82 mmol) in 56% yield. C₄₉H₈₇NO₃ HRMS M+H expected 740.6921; found740.6898 amu. ¹H NMR (500 MHz, CDCl₃) δ 5.41-5.31 (3H, complex); 3.53(2H, dd, J=5.6, 9.9 Hz); 3.47 (2H, dd, J=5.6, 9.9 Hz); 3.44 (2H,multiplet); 3.41 (2H, t, J=6.9 Hz); 3.40 (2H, t, J=6.9 Hz); 3.12 (1H,multiplex); 2.73 (1H, multiplet); 2.38 (6H,s); 2.35 (1H, ddd, J=2.2,4.8, 13.1 Hz); 0.84-2.25 (64 H, complex); 0.68 (3H, s) ppm.

Compounds 5-11 and 13-17 are novel cationic lipids. Compound 12 isS-Octyl CLinDMA. The Compounds can be prepared according to the Schemeabove. The synthesis of Compound 9 is shown below.

Preparation of(2S)-1-({6-[(33)-cholest-5-en-3-yloxylhexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxylpropan-2-amine. (Compound 9)

To a stirred, chilled (−7° C.) solution of the alcohol (41.99 g, 503mmol) and lutidine (5.90 g, 55.1 mmol) in anhydrous CH₂Cl₂ (420 mL) wasadded triflic anhydride (9 mL, 53.3 mmol) portion wise over 30 minutes.The solution was stirred for 2.5 hours, and then transferred to astirred cooled (2° C.) 2M NHMe₂ in THF (1425 mL, 2850 mmol). Thesolution was then stirred for 3 hours. Evaporated volatiles anddissolved oily residue in hexanes (500 mL) and washed with 10% Na₂CO₃(500 mL). The phases separated and aqueous phase back extracted withhexanes (200 mL). The organic phases were combined and washed with water(200 mL), and dried organic over Na₂SO₄. The solution filtered andevaporate volatiles and purified crude through HPFC to afford compound 9(25.5 g, 30.4 mmol) in 60% yield. C₅₆H₁₀₃NO₃ HRMS (ESI positive) M+H,theory m/z 838.8017 amu, measured m/z 838.7996 amu. ¹H NMR (400 MHz,CDCl₃) δ 5.36-5.33 (3H, complex); 4.13-3.40 (10H, complex); 3.13 (1H,multiplet); 2.76 (1H, multiplet); 2.40 (6H, s) 2.35, (1H, ddd, J=2.3,4.8, 13.2 Hz); 2.18 (1H, complex); 2.06-0.86 (77H, complex); 0.68 (3H,s)ppm.

Compound 5: (2 R )-1-{4-[(3 β)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]- N,N-dimethylpropan-2-amine.

-   2.70 g, 3.90 mmol, 57% yield. C₄₆H₈₃NO₃: HRMS (ESI positive) M+H,    theory m/z 698.6446, measured m/z 698.6480. ¹H NMR (500 MHz, CDCl₃)    δ 5.42-5.31, (3H, complex); 3.57-3.38, (10H, complex); 3.12 (1H,    multiplet); 2.73, (1H, multiplet); 2.38 (6H, s); 2.35, (1H, ddd,    J=2.3, 4.8, 13.2 Hz); 2.22-0.84 (58H, complex); 0.68, (3H, s) ppm.

Compound 6: 1-[(2 R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine

-   1.28 g, 1.87 mmol, 51% yield, C₄₃H₇₉N₃O₃: HRMS (ESI positive) M+H,    theory m/z 686.6194 amu, measured m/z 686.6201 amu. ¹H NMR (500 MHz,    CDCl₃) δ 7.92 (1H, d, J=5.9 Hz); 7.52 (2H, br); 7.13 (2H, br); 5.34    (1H, complex); 3.71-3.40 (10H, complex); 3.12 (1H, complex); (2.35    (1H, ddd, 2.2, 4.6, 13.4 Hz); 2.18 (1H, complex); 2.03-0.82 (57H);    0.68 (3H,s) ppm.

Compound 7: 1-[(2 R )-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9 Z, 12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine.

-   2.00 g, 2.35 mmol, 64% yield. C₅₇H₁₀₃N₃: HRMS (ESI positive) M+H,    theory m/z 850.8011 amu, measured m/z 850.8011 amu. ¹H NMR (500 MHz,    CDCl₃) δ 5.42-5.29 (5H, complex); 3.53 (2H, dd, J=5.6, 9.9 Hz); 3.47    (2H, dd, J=5.6, 9.9 Hz); 3.44 (2H, multiplet); 3.40 (2H, t, 6.6 Hz);    3.39 (2H, t, 6.6 Hz); 3.12 (1H, multiplet); 2.77 (2H, t, 6.7 Hz);    2.71 (1H, multiplet); 2.37 (6H, s); 2.35 (1H, ddd, J=2.3, 4.6, 13.2    Hz); 2.18 (1H, multiplet); 2.08-1.93 (6H, complex); 1.92-1.78 (3H,    complex); 1.70-0.84 (66H, complex); 0.68 (3H, s) ppm.

Compound 8: 1-[(2 R )-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9 Z, 12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine.

-   2.90 g, 3.60 mmol, 56% yield. C₅₄H₉₇NO₃: HRMS (ESI positive) M+H,    theory m/z 808.7654 amu, measured m/z 808.7580 amu. ¹H NMR (500 MHz,    CDCl₃) δ 5.42-5.30 (3H complex); 3.56-3.37 (10H, complex); 3.12 (1H,    complex); 2.77 (2H, t, J=6.5 Hz); 2.72 (1H, multiplet); 2.38 (6H,s);    2.35 (1H, ddd, J=2.3, 4.5, 13.4 Hz); 2.26-0.83 (70H, complex); 0.68    (3H,s) ppm.

Compound 9:(2S)-1-({6-[(3β))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine.

-   14.2 g, 16.9 mmol. 68% yield. C₅₆H₁₀₃NO₃ HRMS (ESI positive) M+H,    theory m/z 838.8017 amu, measured m/z 838.7996 amu. ¹H NMR (400 MHz,    CDCl₃) δ 5.36-5.33 (3H, complex); 4.13-3.40 (10H, complex); 3.13    (1H, multiplet); 2.76 (1H, multiplet); 2.40 (6H, s) 2.35, (1H, ddd,    J=2.3, 4.8, 13.2 Hz); 2.18 (1H, complex); 2.06-0.86 (77H, complex);    0.68 (3H,s) ppm.

Compound 10:(3β)-3-[6-{[(2S)-3-[(9Z)-octadec-9-en-1-yloxyl]-2-(pyrrolidin-1-yl)propyl]oxy}hexyl)oxy]cholest-5-ene.

-   1.72 g, 1.99 mmol,81% yield C₅₈H₁₀₅NO₄ HRMS (ESI positive) M+H,    theory m/z 864.8172 amu, measured m/z 864. 8240 amu. ¹H NMR (500    MHz, CDCl₃) δ 5.38-5.30 (3H, complex); 3.58-3.52 (4H, complex);    3.48-3.39 (6H, complex); 3.12 (1H, multiplet); 2.66 (4H, br); 2.50    (1H, q, J=4 Hz); 2.36 (1H, ddd, J=2, 4, 12 Hz) 2.18 (1H, multiplet)    2.05-1.75 (14H, complex); 1.57-0.86 (66H, complex); 0.68 (3H,s) ppm.

Compound 11:(2R)-1-{4-[(3β)-cholest-5-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine.

-   3.79 g. 5.88 mmol, 76% yield. C₄₂H₇₇NO₄₃ HRMS (ESI positive) M+H,    theory m/z 644.5976 amu, measured m/z 644.6012 amu. ¹H NMR(400 MHz,    CDCl₃) δ 5.73 (2H, br); 5.29 (1H, multiplet); 3.56-3.35 (10H,    complex); 3.08 (1H, multiplet); 2.29 (1H, multiplet); 2.13 (1H,    multiplet); 2.01-1.74 (4H, complex); 1.64-0.81 (54H, complex); 0.64    (3H, s) ppm.

Compound 13:(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine.

-   C₄₅H₈₃NO₃ HRMS (ESI positive) M+H, theory m/z 686.6446 amu, measured    m/z 686.6443 amu. ¹H NMR (400 MHz, CDCl₃) δ 5.30 (1H, multiplet);    3.50-3.33 (10H, complex); 3.06 (1H, multiplet); 2.66 (1H,    multiplet); 2.31 (6H, singlet); 2.29 (1H, multiplet); 2.13 (1H,    multiplet); 1.94-1.72 (5H, complex); 1.53-0.80 (54H, complex); 0.62    (3H, singlet) ppm.

Compound 14:(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine.

-   C₄₇H₈₇NO₃ HRMS (ESI positive) M+H, theory m/z 714.6759 amu, measured    m/z 714.6746 amu. ¹H NMR (400 MHz, CDCl₃) δ 5.30 (1H, multiplet);    3.52-3.35 (10H, complex); 3.08 (1H, multiplet); 2.67 (1H,    multiplet); 2.33 (6H, singlet); 2.30 (1H, multiplet); 2.15 (1H,    multiplet); 2.00-1.74 (5H, complex); 1.59-0.82 (58H, complex); 0.64    (3H, singlet) ppm.

Compound 15:(2R)-1-({8-[(3β)-cholest-5-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1-yloxy]propan-2-amine.

-   C₄₅H₈₁NO₃ HRMS (ESI positive) M+H, theory m/z 684.6289 amu, measured    m/z 684.6276 amu. ¹H NMR (400 MHz, CDCl₃) δ 5.49 (2H, multiplet);    5.30 (1H, multiplet); 3.99 (2H, d, J=6 Hz); 3.51-3.33 (8H, complex);    3.08 (1H, multiplet); 2.69 (1H, multiplet); 2.33 (6H, singlet); 2.30    (1H, multiplet); 2.20-1.70 (8H, complex); 1.51-0.82 (48H, complex);    0.64 (3H, singlet) ppm.

Compound 16: (2S)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine.

-   C₄₄H₈₁NO₃ HRMS (ESI positive) M+H, theory m/z 672.6289 amu, measured    m/z 672.6285 amu. ¹H NMR (400 MHz, CDCl₃) δ 5.31 (1H, multiplet);    3.51-3.34 (10H, complex); 3.08 (1H, multiplet); 2.67 (1H,    multiplet); 2.33 (6H, singlet); 2.30 (1H, multiplet); 2.14 (1H,    multiplet); 2.00-1.75 (5H, complex); 1.54-0.81 (52H, complex); 0.64    (3H, singlet) ppm.

Compound 17:(2S-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy-3-[2,2,3,3,4,4.5,5,6,6,7,7,8,8.9,9-hexadecafluorononyl)oxyl]N,N-dimethylpropan-2-amine.

-   C₄₉H₇₅F₁₆NO₃ HRMS(ESI positive) M+H, theory m/z 1030.5564 amu,    measured m/z 1030.5506 amu. ¹H NMR (500 MHz, CDCl₃) δ 6.15-5.89 (1H,    tt, J=6.3, 65.0 Hz); 5.30 (1 H, multiplet); 3.95-3.88 (2H, t, J=16.8    Hz); 3.69 (2H, multiplet); 3.51-3.33 (6H, complex); 3.08 (1H,    multiplet); 2.72 (1H, multiplet); 2.33 (6H, singlet); 2.03 (1H,    multiplet); 2.15 (1H, multiplet); 1.96 (2H, multiplet); 1.80 (3H,    multiplet); 1.56-0.82 (45H, complex); 0.64 (3H, singlet) ppm.-   ¹⁹FNMR (500 MHz, CDCl₃) δ−119.92 (2F, singlet); −122.40 (6F,    singlet); −123.73 (4F, singlet); −129.74 (2F, singlet); −137.33 to    −137.44 (2F, doublet, J=55.0 Hz) ppm.

Preparation of2-amino-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propane-1,3-diol.(Compound 18)

To a stirred solution of sodium hydride (2.2 g, 55 mmol) in toluene (200ml) at 0° C. was added 2-amino-2-(hydroxymethyl)propane-1,3-diol (5 g,41.3 mmol) slowly. The resulting slurry was stirred at 0° C. for 1 hour.To this mixture was added linoleyl mesylate (15 g, 43.5 mmol) and theresulting mixture was stirred for 18 hours at ambient temperature. Thereaction was quenched by slow addition of isopropyl alcohol followed byice. The reaction was partitioned between ether and water. The organicswere washed with water and brine, dried over magnesium sulfate; filteredand evaporated in vacuo. The title compound was purified by flashchromatography (0-20% MeOH/DCM-ammonia) to give 3.25 g of desiredproduct. ¹H NMR (400 MHz, CDCl₃) δ 5.39 (m, 4H), 3.53 (s, 4H), 3.49 (s,2H), 3.43 (t, J=6.59 Hz, 2H), 3.41 (s, 2H), 2.78 (t,J=6.4 Hz, 2H), 2.05(m, 6H), 1.72 (bs, 4H), 1.57 (m, 4H), 1.34 (m, 14H),0.89 (t, J=6.78 Hz,3H).

Preparation of2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5en-3-yloxy]nonyl}oxy)-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol. (Compound 19)

To a stirred solution of of2-amino-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propane-1,3-diol(2.7 g, 7.31 mmol) in toluene (50 mL) at 0° C. was added sodium hydride(0.44 g, 11 mmol) in small portions. The resulting slurry was aged for30 minutes and treated with9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]nonyl methanesulfonate(6.5 g, 11 mmol). The reaction was heated to reflux for 24 hours, thencooled to 0° C. and quenched with water. The reaction was partitionedbetween water/ethyl acetate and the organics were washed with water andbrine, dried over sodium sulfate, filtered and evaporated in vacuo. Thecrude material was purified by flash chromatography (0-10% EtOH/EtOAc)to provide 0.91 g (14%) of title compound as a colorless oil. ¹H NMR(400 MHz, CDCl₃) δ 5.36 (m, 4H), 3.50 (bd, 2H, 3.42 (m, 8H), 3.15 (m,1H), 2.85 (m, 1H)( 2.77 (t, J=6.59 Hz, 2H), 2.38 (m, 1H), 2.18 (m, 1H),2.10-1.80 (complex, 10H), 1.60-0.80 (complex, 67H), 0.68 (s, 3H); HRMS(ESI positive) M+H, theory m/z 866.7960 amu, measured m/z 866.7981 amu.

Preparation of2-amino-3-({6-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]hexyl}oxy)-2-{[9Z)-octadec-9-en-1-yloxy]methyl}propan-1ol.(Compound 20)

Compound 20 was prepared according to general Scheme 4 as described forcompound 19. HRMS (ESI positive) M+H, theory m/z 840.7803 amu, measuredm/z 840.7808 amu. ¹H NMR (400 MHz, CDCl₃) δ 5.37 (m, 2H), 3.42 (m, 10H),3.12 (m, 1H), 2.83 (bs. 1H), 2.35 (m, 1H), 2.18 (m, 1H), 2.05-1.78(complex, 10H), 1.55-0.85 (complex, 69H), 0.68 (s, 3H). ADDITIONALINFORMATION

General Scheme 1, described above, depicts the reaction of linoleylalkoxide, formed under the conditions stated, and epichlorohydrin. Ouroriginal understanding of this reaction was that it occurred via a SN2mechanism at the carbon bearing the halide atom with no change at theasymmetric carbon centre, for example, as depicted in the synthesis ofCompound 1. Subsequent chemical modifications of Compound 1, asdepicted, lead to Compound 4 with the stereochemistry unchanged at thatposition.

However, recent studies (experiments on the reaction of lineolyl alcoholwith S-epichlorohydrin, under conditions described, Lewis acidconditions and in conjunction with vibrational circular dichroism (VCD)experiments, a valid spectroscopic method for determining absoluteconfiguration of chiral molecules (R. K. Dukor and L. A. Nafie, inEncyclopedia of Analytical Chemistry: Instrumentation and Applications,Ed. R. A. Meyers (Wiley, Chichester, 2000) 662-676.)), have revealed thereaction to occur via a SN2′ mechanism ie reaction at the terminalcarbon of the epoxide leading to subsequent ring opening, followed by anin situ ring closing step that leads to inversion of stereochemistry atthe asymmetric carbon.

The consequence of this finding is that the stereochemistry in Compounds1, 3,4-11, and 13-16 are incorrectly represented at the asymmetriccarbon and the correct representation to be one where the bond isinverted. For example, Compound 1 should be drawn as shown in 1a; 3should be drawn as shown in 3a; and 4 should be drawn as shown in 4a;and so on.

To be clear, Compounds 1a, 3a, 4a-1a, and 13a-16a were originally madeand tested following the General Schemes above. The studies conducted,as described in Examples 1-3 below and shown in FIGS. 1-4, utilizedCompound 9a, not Compound 9.

-   (2R)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine    (Compound 4a);-   (2S    )-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine    (Compound 5a);-   1-[(2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine    (Compound 6a);-   1-[(2S )-1-{7-[(3β)-cholest-5-en    -3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,    12Z)octadeca-9,12-dien-1-yloxy]propan-2-amine (Compound 7a);-   1-[(2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,    12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine (Compound 8a);-   (2R)-1-({6-[(3β))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N,-dimethyl-3[(9Z)-octadec-9-en-1-yloxy]propan-2-amine    (Compound 9a);-   (3β)-3-[6-{[(2R)-3-[(9Z)-octadec-9-en-1-yloxyl]-2-(pyrrolidin-1-yl)propyl]oxy}hexyl)oxy]cholest-5-ene    (Compound 10a);-   (2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine    (Compound 11a);-   (2S)-1-({8-[(3β)cholest-5en-3-yloxy]octyl}oxy)-N,N-dimenthyl-3-(pentyloxy)propan-2-amine    (Compound 13a);-   (2S)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3(heptyloxy)-N,N-dimethylpropan-2-amine    (Compound 14a);-   (2S)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1-yloxy]propan-2-amine    (Compound 15a); and-   (2R)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine    (Compound 16a)

LNP COMPOSITIONS

LNP Process Description 1:

The Lipid Nano-Particles (LNP) are prepared by an impinging jet process.The particles are formed by mixing equal volumes of lipids dissolved inalcohol with siRNA dissolved in a citrate buffer. The lipid solutioncontains a novel cationic lipid of the instant invention, a helper lipid(cholesterol) and PEG (PEG-DMG) lipid at a concentration of 5-15 mg/mLwith a target of 9-12 mg/mL in an alcohol (for example ethanol). Theratio of the lipids has a mole percent range of 25-98 for the cationiclipid with a target of 45-65, the helper lipid has a mole percent rangefrom 0-75 with a target of 30-50 and the PEG lipid has a mole percentrange from 1 -6 with a target of 2-5. The siRNA solution contains one ormore siRNA sequences at a concentration range from 0.7 to 1 .0 mg/mLwith a target of 0.8 -0.9 mg/mL in a sodium citrate: sodium chloridebuffer pH 4. The two liquids are mixed in an impinging jet mixerinstantly forming the LNP. The teeID has a range from 0.25 to 1.0 mm anda total flow rate from 10 -200 mL/min. The combination of flow rate andtubing ID has effect of controlling the particle size of the LNPsbetween 50 and 200 nm. The mixed LNPs are held from 30 minutes to 48 hrsprior to a dilution step. The dilution step comprises similar impingingjet mixing which instantly dilutes the LNP. This process uses tubing IDsranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 400 mL/min.The LNPs are concentrated and diafiltered via an ultrafiltration processwhere the alcohol is removed and the citrate buffer is exchanged for thefinal buffer solution such as phosphate buffered saline. Theultrafiltration process uses a tangential flow filtration format (TFF).This process uses a membrane nominal molecular weight cutoff range from30-500 KD. The membrane format can be hollow fiber or flat sheetcassette. The TFF processes with the proper molecular weight cutoffretains the LNP in the retentate and the filtrate or permeate containsthe alcohol; citrate buffer; final buffer wastes. The TFF process is amultiple step process with an initial concentration to a siRNAconcentration of 1-3 mg/mL. Following concentration, the LNPs solutionis diafiltered against the final buffer for 15 -20 volumes to remove thealcohol and perform buffer exchange. The material is then concentratedan additional 1-3 fold. The final steps of the LNP process are tosterile filter the concentrated LNP solution and vial the product.

Analytical Procedure:

1) siRNA Concentration

The siRNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), are treated with 0.5% Triton X-100 to free total siRNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with liner gradient from 0-15 min in and flow rate of 1ml/min. The siRNA amount is determined by comparing to the siRNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 are used to determine the free siRNA and total siRNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples are excited at 485nm and fluorescence emission was measured at 530 nm. The siRNA amount isdetermined by comparing to the siRNA standard curve.Encapsulation rate=(1-free siRNA/total siRNA)×100%3) Particle Size and Polydispersity

RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with1×PBS. The particle size and polydispersity of the samples is measuredby a dynamic light scattering method using ZetaPALS instrument(Brookhaven Instruments Corporation, Holtsville, N.Y.). The scatteredintensity is measured with He—Ne laser at 25° C. with a scattering angleof 90°.

4) Zeta Potential Analysis

RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials.

5) Lipid Analysis

Individual lipid concentrations are determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc. Chelmsford, Mass.).Individual lipids in RDVs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient is 75% mobile phase A and 25% mobile phase B from time 0 to0.10 min; 25% mobile phase A and 75% mobile phase B from 0.10 to 1.10min; 25% mobile phase A and 75% mobile phase B from 1.10 to 5.60 min; 3%mobile phase A and 95% mobile phase B from 5.60 to 8.01 min; and 75%mobile phase A and 25% mobile phase B from 8.01 to 13 min with flow rateof 1 ml/min. The individual lipid concentration is determined bycomparing to the standard curve with all the lipid components in theRDVs with a quadratic curve fit. The molar percentage of each lipid iscalculated based on its molecular weight.

Utilizing the above described LNP process, specific LNPs with thefollowing ratios and siRNAs were identified:

Nominal Composition:

Cationic Lipid/Cholesterol/PEG-DMG 60/38/2. Luc siRNA (SEQ. ID. NO.: 1)5′-iB-A U AAGG CU A U GAAGAGA U ATT-iB 3′ (SEQ. ID. NO.: 2)3′-UUUAUUCCGAUACUUCUC UAU-5′ AUGC - Ribose iB-Inverted deoxy abasicUC - 2′ Fluoro AGT - 2′ Deoxy AGU - 2′ OCH₃Cationic Lipid/Cholesterol/PEG-DMG 60/38/2 ApoB siRNA (SEQ ID NO.: 3)5′-iB-CUUU AACAA UUCCU GAAA U TT-iB (SEQ ID NO.: 4)3′-UUGAAAUUGUUAAGGACU UUA-5′ AUGC - Ribose iB - Inverted deoxy abasicUC - 2′ Fluoro AGT - 2′ Deoxy AGU - 2′ OCH₃LNP Process Description 2:

The Lipid Nano-Particles (LNP) are prepared by an impinging jet process.The particles are formed by mixing lipids dissolved in alcohol withsiRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNAare targeted at 45-55% lipid and 65-45% siRNA. The lipid solutioncontains a novel cationic lipid of the instant invention, a helper lipid(cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at aconcentration of 5-15 mg/mL with a target of 9-12 mg/mL in an alcohol(for example ethanol). The ratio of the lipids has a mole percent rangeof 25-98 for the cationic lipid with a target of 35-65, the helper lipidhas a mole percent range from 0-75 with a target of 30-50, the PEG lipidhas a mole percent range from 1-15 with a target of 1-6, and the DSPChas a mole precent range of 0-15 with a target of 0-12. The siRNAsolution contains one or more siRNA sequences at a concentration rangefrom 0.3 to 1 .0 mg/mL with a target of 0.3 -0.9 mg/mL in asodium-citrate buffered salt solution with pH in the range of 3.5-5. Thetwo liquids are heated to a temperature in the range of 15-40° C.,targeting 30-40° C., and then mixed in an impinging jet mixer instantlyforming the LNP. The teeID has a range from 0.25 to 1.0 mm and a totalflow rate from 10 -600 mL/min. The combination of flow rate and tubingID has effect of controlling the particle size of the LNPs between 30and 200 nm. The solution is then mixed with a buffered solution at ahigher pH with a mixing ratio in the range of 1:1 to 1:3 vol:vol buttargeting 1:2 vol:vol. This buffered solution is at a temperature in therange of 15-40° C., targeting 30-40° C. The mixed LNPs are held from 30minutes to 2 hrs prior to an anion exchange filtration step. Thetemperature during incubating is in the range of 15-40° C., targeting30-40° C. After incubating the solution is filtered through a 0.8 umfilter containing an anion exchange separation step. This process usestubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to2000 mL/min. The LNPs are concentrated and diafiltered via anultrafiltration process where the alcohol is removed and the citratebuffer is exchanged for the final buffer solution such as phosphatebuffered saline. The ultrafiltration process uses a tangential flowfiltration format (TFF). This process uses a membrane nominal molecularweight cutoff range from 30-500 KD. The membrane format can be hollowfiber or flat sheet cassette. The TFF processes with the propermolecular weight cutoff retains the LNP in the retentate and thefiltrate or permeate contains the alcohol; citrate buffer; final bufferwastes. The TFF process is a multiple step process with an initialconcentration to a siRNA concentration of 1-3 mg/mL. Followingconcentration, the LNPs solution is diafiltered against the final bufferfor 10-20 volumes to remove the alcohol and perform buffer exchange. Thematerial is then concentrated an additional 1-3 fold. The final steps ofthe LNP process are to sterile filter the concentrated LNP solution andvial the product.

EXAMPLE 1

Mouse In Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing Compound 9 or 12, in the nominal compositions describedimmediately above, were evaluated for in vivo efficacy and induction ofinflammatory cytokines in a luciferase mouse model. The siRNA targetsthe mRNA transcript for the firefly (Photinus pyralis) luciferase gene(Accession # M15077). The primary sequence and chemical modificationpattern of the luciferase siRNA is displayed above. The in vivoluciferase model employs a transgenic mouse in which the fireflyluciferase coding sequence is present in all cells.ROSA26-LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from theDana Farber Cancer Institute are induced to express the Luciferase geneby first removing the LSL sequence with a recombinant Ad-Cre virus(Vector Biolabs). Due to the organo-tropic nature of the virus,expression is limited to the liver when delivered via tail veininjection. Luciferase expression levels in liver are quantitated bymeasuring light output, using an IVIS imager (Xenogen) followingadministration of the luciferin substrate (Caliper Life Sciences).Pre-dose luminescence levels are measured prior to administration of theRDVs. Luciferin in PBS (15 mg/mL) is intraperitoneally (IP) injected ina volume of 150 uL. After a four minute incubation period mice areanesthetized with isoflurane and placed in the IVIS. imager. The RDVs(containing siRNA) in PBS vehicle were tail vein injected in a volume of0.2 mL. Final dose levels ranged from 0.3 to 3 mg/kg siRNA. PBS vehiclealone was dosed as a control. Three hours post dose, mice were bledretro-orbitally to obtain plasma for cytokine analysis. Mice were imaged48 hours post dose using the method described above. Changes inluciferin light output directly correlate with luciferase mRNA levelsand represent an indirect measure of luciferase siRNA activity. In vivoefficacy results are expressed as % inhibition of luminescence relativeto pre-dose luminescence levels. Plasma cytokine levels were determinedusing the SearchLight multiplexed cytokine chemoluminescent array(Pierce/Thermo). Systemic administration of the luciferase siRNA RDVsdecreased luciferase expression in a dose dependant manner. Greaterefficacy was observed in mice dosed with compounds 9 containing RDVsthan with the RDV containing the octyl-CLinDMA cationic lipid. Compound12, (Table 1 and FIG. 2). Compound 9 and 12 RDVs significantly increasedmouse plasma levels of the cytokines IL-6 and mKC relative to the PBScontrol. However, average cytokine induction was lower in the animalsdosed with the Compound 9 RDV, relative to the Compound 12 RDV.(FIG. 1).

TABLE 1 Mouse In Vivo efficacy data. Average % Inhibition ofBioluminescence by LNPs prepared from compounds 9 and 12 Compound 9Compound 12 0.1 mg 0.3 mg 1.0 mg 0.1 mg 0.3 mg 1.0 mg Kg⁻¹ Kg⁻¹ Kg⁻¹Kg⁻¹ Kg⁻¹ Kg⁻¹ 80 89 91 54 74 88

EXAMPLE 2

Rat In Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing Compounds 9 or 12 in the nominal compositions describedabove, were evaluated for in vivo efficacy and increases in alanineamino transferase and aspartate amino transferase in Sprague-Dawley(Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNAtranscript for the ApoB gene (Accession # NM 019287). The primarysequence and chemical modification pattern of the ApoB siRNA isdisplayed above. The-RDVs (containing siRNA) in PBS vehicle were tailvein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately3 ml/min. Five rats were used in each dosing group. After LNPadministration, rats are placed in cages with normal diet and waterpresent. Six hours post dose, food is removed from the cages. Animalnecropsy is performed 24 hours after LNP dosing. Rats are anesthetizedunder isoflurane for 5 minutes, then maintained under anesthesia byplacing them in nose cones continuing the delivery of isoflurane untilex-sanguination is completed. Blood is collected from the vena cavausing a 23 guage butterfly venipuncture set and aliquoted to serumseparator vacutainers for serum chemistry analysis. Punches of theexcised caudate liver lobe are taken and placed in RNA Later (Ambion)for mRNA analysis. Preserved liver tissue was homogenized and total RNAisolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNAisolation kit following the manufacturer's instructions. Liver ApoB mRNAlevels were determined by quantitative RT-PCR. Message was amplifiedfrom purified RNA utilizing a rat ApoB commercial probe set (AppliedBiosystems Cat # RN01499054_ml). The PCR reaction was performed on anABI 7500 instrument with a 96-well Fast Block. The ApoB mRNA level isnormalized to the housekeeping PPIB (NM 011149) mRNA. PPIB mRNA levelswere determined by RT-PCR using a commercial probe set (AppliedBiosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio ofApoB mRNA/PPIB mRNA. All mRNA data is expressed relative to the PBScontrol dose. Serum ALT and AST analysis were performed on the SiemensAdvia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanineaminotransferase (Cat# 03039631) and aspartate aminotransferase (Cat#03039631) reagents. The RDVs employing both Compounds 9 and 12 displayedsimilar levels of ApoB mRNA knock down at the 1 mpk dose (FIG. 3). Anincrease in LFTs were observed for both RDVs, relative to PBS controldosed rats (FIG. 3). While ALT levels increased to a greater extent inrats dosed with the Compound 9 RDV, AST levels increased more stronglywith the Compound 12 RDV (FIG. 3).

EXAMPLE 3

Rhesus Monkey In Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing Compounds 9 or 12 in the nominal compositions describedabove, were evaluated for in vivo efficacy and increases in alanineamino transferases (ALT) and aspartate amino transferase (AST) in maleor female Macaca mulatta (rhesus) monkeys. The siRNA targets the mRNAtranscript for the ApoB gene (Accession # XM 001097404). The primarysequence and chemical modification pattern of the ApoB siRNA isdisplayed above. The RDVs (containing siRNA) in PBS vehicle wereadministered by intravenous injection in the saphenous vein at aninjection rate of 20 mL/minute to a dose level of 3.6 mg/kilogram siRNA.The injection volumes were from 1.9 to 2.1 mL/kilogram and monkeysranged in weight from 2.5 to 4.5 kilograms. Each RDV or PBS control wasadministered to two monkeys. At multiple days post dose, 1 mL bloodsamples were drawn from the femoral artery for serum chemistry analysis.Monkeys were fasted overnight prior to blood draws. Serum ALT and ASTanalysis were performed on the Siemens Advia 1800 Clinical ChemistryAnalyzer utilizing the Siemens alanine aminotransferase (Cat# 03039631)and aspartate aminotransferase (Cat# 03039631) reagents. As a measure ofefficacy, LDL-C was monitored as a downstream surrogate marker of ApoBmRNA reduction. At eight days post systemic administration, of RDVscontaining Compounds 9 or 12, reduced serum levels of LDL-C to less than50% of pre-dose levels (FIG. 4). The RDV containing Compound 9 reducedLDL-C levels to a greater extent than the RDV containing Compound 12(ave.: −63% vs. −53%). ALT and AST were elevated relative to predosevalues in monkeys treated with both Compounds 9 and 12 RDVs (FIG. 4).Elevation of ALT was approximately equivalent for both RDVs, but AST waselevated to a markedly higher level in the RDV containing Compound 12.

What is claimed is:
 1. A lipid nanoparticle comprising a cationic lipidof Formula A:

wherein: p is 1 to 8; R¹ and R² are independently selected from H,(C₁-C₁₀)alkyl, heterocyclyl, and a polyamine, wherein said heterocyclyland polyamine are optionally substituted with one to three substituentsselected from R⁴, or R¹ and R² can be taken together with the nitrogento which they are attached to form a monocyclic heterocycle with 4-7members optionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle optionally substituted with one to three substituentsselected from R⁴; R³ is selected from H and (C₁-C₆)alkyl, said alkyloptionally substituted with one to three substituents selected from R⁴;R⁴ is independently selected from halogen, OR⁵, SR⁵, CN, CO₂R⁵ andCON(R⁵)₂; R⁵ is independently selected from H, (C₁-C₁₀)alkyl and aryl;and Y is a (C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl, or a (C₄-C₂₂)alkenyl;or any pharmaceutically acceptable salt or stereoisomer thereof.
 2. Thelipid nanoparticle of claim 1, wherein: p is 1 to 8;

is selected from:

R³ is H; and Y is a (C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl, or a(C₄-C₂₂)alkenyl; or any pharmaceutically acceptable salt or stereoisomerthereof.
 3. The lipid nanoparticle of claim 1, wherein the cationiclipid is selected from:(2S)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine(Compound 4);(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine(Compound 5);(2R)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 7);(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 8); (2 S)-1 -({6-[(3β)-cholest-5 -en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine(Compound 9);(3β)-3-[6-{[(2S)-3-[(9Z)-octadec-9-en-1-yloxyl]-2-(pyrrolidin-1-yl)propyl]oxy}hexyloxy]cholest-5-ene(Compound 10);(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine(Compound 11);(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine(Compound 13);(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine(Compound 14);(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1-yloxy]propan-2-amine(Compound 15);(2S)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine(Compound 16);(2S)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-[2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorononyl]oxy-N,N-dimethylpropan-2-amine(Compound17);2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]nonyl}oxy)-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 19); and;2-amino-3-({6-[(3β,8ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]hexyl}oxy)-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 20); or anypharmaceutically acceptable salt or stereoisomer thereof.
 4. The lipidnanoparticle of claim 1, wherein the cationic lipid is selected from:(2R)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine(Compound 4a);(2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine(Compound 5a);(2S)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 7a);(2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine(Compound 8a);(2R)-1-({6-[(3β)-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine(Compound 9a);(3β)-3-[6-{[(2R)-3-[(9Z)-octadec-9-en-1-yloxyl]-2-(pyrrolidin-1-yl)propyl]oxy}hexyloxy]cholest-5-ene(Compound 10a);(2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine(Compound 11a);(2S)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine(Compound13a);(2S)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine(Compound14a);(2R)-1-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1-yloxy]propan-2-amine(Compound15a); and(2R)-1-butoxy-3-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine(Compound16a).
 5. The lipid nanoparticle of claim 1, wherein the cationic lipidis:(2S)-1-({6-[(3β)-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine(Compound9); or(2R)-1-({6-[(3β)-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine(Compound 9a) or any pharmaceutically acceptable salt or stereoisomerthereof.
 6. The lipid nanoparticle of claim 1, wherein the nanoparticlefurther comprises an oligonucleotide.
 7. The lipid nanoparticle of claim6, wherein the oligonucleotide is siRNA or miRNA.
 8. The lipidnanoparticle of claim 7, wherein the oligonucleotide is siRNA.
 9. Alipid nanoparticle comprising a cationic lipid of Formula A:

wherein: p is 1 to 8;

 is selected from:

n is 1 to 10; R³ is H; and Y is a (C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl,or a (C₄-C₂₂)alkenyl; or any pharmaceutically acceptable salt orstereoisomer thereof.
 10. The lipid nanoparticle of claim 9, wherein thenanoparticle further comprises an oligonucleotide.
 11. The lipidnanoparticle of claim 10, wherein the oligonucleotide is siRNA or miRNA.12. The lipid nanoparticle of claim 11, wherein the oligonucleotide issiRNA.
 13. A lipid nanoparticle comprising a cationic lipid of FormulaA:

wherein: p is 1 to 8;

 is

R³ is H; and Y is a (C₄-C₂₂)alkyl, (C₄-C₂₂)perfluoroalkyl, or a(C₄-C₂₂)alkenyl; or any pharmaceutically acceptable salt or stereoisomerthereof.
 14. The lipid nanoparticle of claim 13, wherein the cationiclipid is:1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine(Compound 6); or1-[(2S)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine.15. The lipid nanoparticle of claim 13, wherein the nanoparticle furthercomprises an oligonucleotide.
 16. The lipid nanoparticle of claim 15,wherein the oligonucleotide is siRNA or miRNA.
 17. The lipidnanoparticle of claim 16, wherein the oligonucleotide is siRNA.